Understanding the role that T cells play in the chronic allergic inflammatory skin disorder, atopic dermatitis (AD), is highly relevant to the development of specific treatments for this debilitating disease. Regulatory T cells (Tregs) normally suppress pro-inflammatory T cells in healthy subjects. The proposed studies will investigate whether Tregs from AD patients can actually contribute to inflammatory responses in the skin. The primary objectives are as follows: (1) To examine the capacity for Tregs to convert to pathogenic T cells which secrete Th2 cytokines upon exposure to factors expressed at the site of allergic inflammation; (2) To determine whether treatments which diminish Th2 pathways can enhance the induction of protective Tregs in vitro. Low expression of CD127 (IL-7R 1 chain) will be used to isolate natural (CD25+CD127lo) and adaptive (CD25negCD127lo) Tregs from AD patients. Data suggests that natural Tregs from these patients can acquire Th2 effector properties, while IL-10-producing adaptive Tregs are protective. In Aim 1, studies will elucidate the properties of distinct CD127lo Treg types, compare their susceptibility to T cell-activating factors expressed in AD skin, and determine their pathogenic versus protective potential. Multi-color flow cytometry and standard in vitro T cell assays will be used to analyze the phenotype and suppressive function of distinct CD127lo Treg types. Responsiveness of CD127lo T cells to Th2-promoting factors expressed in AD skin (thymic stromal lymphopoietin (TSLP), allergen, or bacterial superantigen) will be examined using flow cytometry imaging to identify IL-4+ T cells undergoing mitosis at the single-cell level. The role of transcription factors involved in Th2 polarization and IL-4 receptor signaling (STAT5 and STAT6) will be assessed in Treg activation mediated by TSLP and in amplification of this response by allergen. Small interfering RNAs will be used to test whether inhibiting Th2 signaling molecules in CD25+CD127lo Tregs can block effector function and restore suppressor activity. In Aim 2, the presence of distinct types of CD127lo Tregs at the site of allergic inflammation will first be confirmed in skin biopsies from atopy patch test (APT) sites. The capacity for Tregs to convert to Th2 effectors at these sites will be tested in a novel skin explant assay by analyzing the Th2 effector properties of CD25+CD127lo Tregs cultured with keratinocytes from APT sites. In Aim 3, the goal is to identify treatment strategies which could be used to enhance induction of protective Tregs. The relationship of IL-10-secreting Tregs to other CD127lo Treg types will be analyzed using an allergen variant (H22-Fel d 1) which induces IL-10-expressing T cells. Allergen-specific IL-10-secreting Tregs will be expanded in vitro in order to determine their protective properties and to compare IL-10-secreting Tregs derived from distinct CD127lo precursors. The role of Th2 cytokines in inhibiting induction of IL-10-producing Tregs in vitro will be examined. Changes in induction of this Treg type will be monitored ex vivo during treatments which are known to improve skin condition and to diminish Th2 pathways. PUBLIC HEALTH RELEVANCE It is widely recognized that T cells play an important role in the inflammation that is a cardinal feature of the chronic allergic skin disease, atopic dermatitis (AD). Although considerable progress has been made in defining the role of regulatory T cells (Tregs) in allergic disease, there are still major gaps in our knowledge. The proposed studies, which investigate how Tregs function in AD skin, are highly relevant to the development of T cell-based treatments for this debilitating disease.